Rodent Antibody Isotyping Kits Technical advice and FAQs

Instructions for use

Note: All reagents should be brought to room temperature before use.

Sample preparation:

Dilute all monoclonal antibody samples to a concentration of 1.0 ug/ml in PBS containing 1% w/v bovine serum albumin (BSA). If the concentration of the sample is entirely unknown, make dilutions based on the following estimates:

150ul of the diluted sample will be added to the development tubes.

Assay protocol:

Remove the required number of isotyping strips from the desiccant vial and replace the cap. Remove the caps from an equal number of development tubes. Note: the tubes may be labeled with a marker for identification.

Pipette 150ul of the freshly diluted sample into each development tube. Vortex the tube briefly to ensure that the coloured micro particle solution is completely re-suspended
and then incubate at room temperature for 30 seconds

Place one isotyping strip with the solid red end at the bottom into each development tube.

Interpretation of results:

Interpret the results at 5-10 minutes once the positive flow control bands have appeared. Within 5-10 minutes, a blue band will appear above the letters in one of the class or subclass windows as well as in the kappa window of the strip, indicating the heavy and light-chain composition of the monoclonal antibody. Lambda light chains are very rare in rat monoclonal antibodies, but if present no positive band will be seen in the kappa window. The intensity of the blue bands will increase as the sample continues to flow up the strip. The positive flow control bands on one side of the isotyping test strip should also appear, indicating that the antibody-coated micro particles are functional and have flowed up the strip. In cases where the sample is very dilute, the development time may take up to 10 minutes.

Note: For a permanent experimental record or for an easier interpretation of results when testing multiple samples, the solid red area may be cut off the bottom of the strip to prevent further band development once the positive flow control bands have appeared. A gentle stream of air can be applied to the membrane portion of the strip to assist in drying the membrane and preventing any further development. Do not wash the strip to stop the reaction.

FAQs

Postive flow control bands appeared by no heavy or light chain band

Possible causes:

The antibody concentration was too low - prepare a less dilute sample and re-test.

No antibody was in the sample - the hybridoma is either not secreting or is not a rat monoclonal. If possible sub-clone the hybridoma and re-test.

Freshly diluted samples were not used - prepare fresh dilutions and re-test.

Appearance of multiple heavy and light chain bands on the strip

Possible causes:

Antibody concentration was too high - dilute sample further and re-test

For ascites, there may be small amounts of contaminating antibodies produced - dilute sample further and re-test

For tissue culture supernatant, a mixed culture may be present - re-clone the hybridoma and re-test

No positive flow control bands appear

Possible causes:

Sample volume was too low (<150ul) - carefully dilute a fresh sample and pipette 150ul into a new development tube and re-test

Strip removed from development tube too early - re-test and allow strip to react for at least 10 minutes